[unreadable] Human infections caused by Gram-positive bacteria present a serious therapeutic challenge due to the emergence of antibiotic-resistant strains. Of concern are infections with S. aureus, S. epidermidis, and E. faecalis, microorganisms that are the most common cause of bacterial disease in American hospitals. These Gram-positive human pathogens have acquired resistance mechanisms to virtually all known antibiotics. The development of novel targets for antimicrobial therapy is therefore urgently needed. We are investigating the protein translocation pathways across the cell wall envelope of Gram-positive bacteria as potential targets for antimicrobial therapy. Experiments described in this application aim at identifying S. aureus genes that are required for the secretion of hemolysins, lipase and nuclease, exotoxin that are known play important roles during infection. Two approaches will be used. By generating knockout mutations in the prsA genes of S. aureus, we will ask whether the encoded peptidyl-prolyl isomerases are essential for the secretion of folded polypeptides. By conducting a generalized transposon mutagenesis and scoring for defects in the secretion of exotoxins, we will ask whether we can identify all genes required for S. aureus secretion. The identified genes will be characterized for their role in the establishment of S. aureus infections using a mouse model of disease. If we can identify genes and gene products in the S. aureus secretory pathway that are involved in the establishment of disease, the gene product could represent a novel target for antimicrobial therapy. [unreadable] [unreadable]